Methods of suppressing microglial activation

ABSTRACT

Methods of suppressing the activation of microglial cells in the Central Nervous System (CNS), methods of ameliorating or treating the neurological effects of cerebral ischemia or cerebral inflammation, and methods of combating specific diseases that affect the CNS by administering a compound that binds to microglial receptors and prevents or reduces microglial activation are described. Also described are methods of screening compounds for the ability to suppress or reduce microglial activation.

RELATED APPLICATION INFORMATION

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/260,430, filed Mar. 1, 1999, which in turnclaims the benefit of U.S. Provisional Application No. 60/077,551, filedMar. 11, 1998, the disclosures of both of which are incorporated byreference herein in their entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with Government support under NIH grantsNS368087-01A2, K08NS01949, and RO3 AG16507-01. The Government hascertain rights to this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to method of suppressing theactivation of microglial cells in the Central Nervous System (CNS),methods of reducing or suppressing the activation of glial or microglialcells, methods of ameliorating or treating the neurological effects ofcerebral ischemia or cerebral inflammation, methods of combatingspecific diseases that affect the CNS by administering a compound thatbinds to microglial receptors and prevents or reduces microglialactivation, and methods of screening compounds for the ability toprevent or reduce microglial activation.

BACKGROUND OF THE INVENTION

[0004] The Central Nervous System (CNS) has long been considered to be asite of relative immune privilege. However, it is increasinglyrecognized that CNS tissue injury in acute and chronic neurologicaldisease may be mediated by the CNS inflammatory response. The CNSinflammatory response is primarily mediated by inflammatory cytokines.

[0005] Apolipoprotein E (ApoE) is a 299 amino acid lipid-carryingprotein with a known sequence (Rall et al., J. Biol. Chem. 257:4174(1982); McLean et al., J. Biol. Chem. 259:6498 (1984). The complete genefor human ApoE has also been sequenced (Paik et al., Proc. Natl. Acad.Sci. USA 82:3445 (1985). ApoE sequences from at least ten species havebeen determined, and show a high degree of conservations across species,except at the amino and carboxyl termini. Weisgraber, Advances inProtein Chemistry 45:249 (1994).

[0006] Human ApoE is found in three major isoforms: ApoE2, ApoE3, andApoE4; these isoforms differ by amino acid substitutions at positions112 and 158. The most common isoform is ApoE3, which contains cysteineat residue 112 and arginine at residue 158; ApoE2 is the least commonisoform and contains cysteine at residues 112 and 158; ApoE4 containsarginine at residues 112 and 158. Additional rare sequence mutations ofhuman ApoE are known (see, e.g., Weisgraber, Advances in ProteinChemistry 45:249 (1994),at page 268-269). The presence of ApoE4 has beenassociated with risk of developing sporadic and late-onset Alzheimer'sdisease (Strittmatter et al., Proc. Natl. Acad. Sci. USA 90:1977-1980(1993)).

[0007] ApoE plays a role in cholesterol metabolism and has also beenreported to have immunomodulatory properties. It is secreted bymacrophages after peripheral nerve injury and by astrocytes andoligodendrocytes (glial cells) after Central Nervous System (CNS)injury.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the finding that microglialactivation can be reduced or suppressed using peptides that comprise thereceptor binding site sequence of Apolipoprotein E. Thus, the presentinvention provides methods and compositions for treating CNS diseasestates in which glial or microglial activation occurs, and in whichglial or microglial activation contributes to the deleterious signsand/or symptoms associated with the specific disease state.

[0009] The present invention is further based upon the identification ofthe aforesaid receptor as a high affinity Apolipoprotein E receptor,with the binding characteristics of the LRP/α2M receptor.

[0010] In view of the foregoing, a first aspect of the present inventionis a method of suppressing glial or microglial activation in a mammal byadministering a compound that binds to glial or microglial cells at theLRP/α2M receptor (the receptor bound by a peptide of SEQ ID NO:3 or SEQID NO:6). The compound is administered in an amount that reduces glialor microglial activation compared to activation that which would occurin the absence of the compound.

[0011] A further aspect of the present invention is a method ofameliorating symptoms associated with CNS inflammation by administeringa compound that binds to glial or microglial cells at the LRP/α2Mreceptor (the receptor bound by a peptide of SEQ ID NO:3 or SEQ IDNO:6).

[0012] A further aspect of the present invention is a method ofameliorating symptoms associated with CNS ischemia in a subject, byadministering a compound that binds to glial or microglial cells at theLRP/α2M receptor (the receptor bound by a peptide of SEQ ID NO:3 or SEQID NO:6) in a treatment effective amount.

[0013] A further aspect of the present invention is a method of treatingcerebral ischemia or inflammation of the CNS by administering a LRP/α2Mreceptor ligand, such as peptide comprising SEQ ID NO:3 or SEQ ID NO:6.

[0014] A further aspect of the present invention is a therapeuticpeptide of SEQ ID NO: 3, or a dimer of two peptides wherein each peptidecomprises SEQ ID NO:2, or a peptide of SEQ ID NO:6, and pharmaceuticalcompositions thereof.

[0015] A further aspect of the present invention is a method ofscreening a compound for the ability to suppress glial or microglialactivation by incubating an activated glial or microglial cell culturewith the compound, and then measuring a marker of microglial activationsuch as nitric oxide.

[0016] A further aspect of the present invention is a method ofscreening a compound for the ability to suppress glial or microglialactivation, by pre-incubating a glial or microglial cell culture withthe compound; incubating the cell culture with a known activator of gliaor microglia; and then measuring a marker of glial or microglialactivation.

[0017] A further aspect of the present invention is a method ofscreening a test compound for the ability to suppress glial ormicroglial activation, by determining whether the compound binds to gliaor microglia at the same receptor to which peptides of SEQ ID NO:3 orSEQ ID NO:6 bind (that is, the LRP/α2M receptor).

[0018] A further aspect of the present invention is a method ofsuppressing macrophage activation in a mammalian subject, byadministering a compound that binds to macrophage cells at the LRP/α2Mreceptor (the receptor bound by a peptide of SEQ ID NO:3 or SEQ IDNO:6).

[0019] A further aspect of the present invention is a method of treatingatherosclerosis or of reducing the formation of atherosclerotic plaques,comprising administering a compound that binds to macrophage cells atthe LRP/α2M receptor (the receptor bound by a peptide of SEQ ID NO:3 orSEQ ID NO:6).

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 graphs the production of nitrite by cultures of glial cellsfrom ApoE-deficient mice (solid bar), ApoE3 transgenic mice (hatchedbar), and control mice (white bar), after exposure to lipopolysaccharide(LPS). Responses were measured at 24 and 60 hours after stimulation ofcell cultures by LPS.

[0021]FIG. 2 graphs nitrite production by enriched microglia primarycultures from ApoE-deficient mice after stimulation with LPS andsubsequent addition of peptides of SEQ ID NO:3 (tandem repeat peptides).Peptides were added in doses of from 0 μM to 1000 μM, and a dosedependent decrease in nitrite production was observed. As a control,peptides of SEQ ID NO:2 were added to cultures (solid bar); no decreasein nitrite production was observed.

[0022]FIG. 3A graphs intracellular calcium content over time in murineperitoneal macrophages, after exposure to either ApoE3 (squares) orApoE4 (circles).

[0023]FIG. 3B graphs inositol trisphosphate (IP3) in murine peritonealmacrophages exposed to either ApoE3 (squares) or ApoE4 (circles). Thegraph shows the percent change in IP3 content in treated cells comparedto control cells exposed to vehicle but not ApoE.

[0024]FIG. 4 graphs production of TNFα (picogram/ml) by microgliaprimary cultures from ApoE-deficient mice after addition of peptides ofSEQ ID NO:6 (squares), or addition of peptides of SEQ ID NO:6 and LPS(100 ng/ml) (circles). Peptides were added in doses of 10 μM, 100 μM and1000 μM.

[0025]FIG. 5 is a graph of the optical density of cell cultures, as ameasure of cell viability. Cultures of microglia from ApoE-deficientmice were exposed to either peptides of SEQ ID NO:6 (squares), orpeptides of SEQ ID NO:6 and LPS (100 ng/ml) (circles). Peptides wereadded in doses of 10 μM, 100 μM and 1000 μM.

[0026]FIG. 6 graphs production of TNFα (picogram/ml) by microgliaprimary cultures from ApoE-deficient mice after addition of peptides ofSEQ ID NO:6 (squares), or addition of peptides of SEQ ID NO:6 and LPS(100 ng/ml) (circles). Peptides were added in doses of 1 μM, 10 μM, 100μM and 1000 μM.

[0027]FIG. 7 is a graph of the optical density of cell cultures, as ameasure of cell viability. Cultures of microglia from ApoE-deficientmice were exposed to either peptides of SEQ ID NO:6 (squares), orpeptides of SEQ ID NO:6 and LPS (100 ng/ml) (circles). Peptides wereadded in doses of 10 μM, 100 μM and 1000 μM.

[0028]FIG. 8. Changes in [Ca²⁺]_(i) in macrophages treated with apoE.Panel A: Changes in [Ca²⁺]_(i) in a single Fura-2/AM loaded peritonealmacrophage on stimulation with apoE (100 pM). Details for measuring[Ca²⁺]_(i) are described in the Examples below. The graph shown isrepresentative of 5 individual experiments using 20-30 cells each.Approximately 70-80% of the macrophage demonstrated changes in[Ca²⁺]_(i) upon stimulation with apoE. The arrow indicates the time ofaddition of apoE. Panel B: Effect of apoE concentration on changes in[Ca²⁺]_(i). The changes in [Ca²⁺]_(i) in individual cells were measuredprior to and following exposure to varying concentrations of apoE. Thedata are displayed as mean ( S.E. and are representative of twoindependent experiments; in each case 25-30 cells were analyzed cellsper study.

[0029]FIG. 9. Changes in IP₃ in macrophages treated with apoE. Panel A:Effect of apoE on IP₃ synthesis in macrophages, and modulation bypertussis toxin. These results are representative of two independentexperiments performed in duplicate and expressed as % change in IP₃formation at different time periods in myo-[2-³H]inositol-labeled cellsstimulated with apoE (100 pM) in the presence (open circles) and absence(filled circles) of pertussis toxin. Panel B: Effect of apoEconcentration on IP₃ formation in [³H]labeled macrophages. The cellswere stimulated with varying concentrations of apoE for 60s and IP₃determined. Results are displayed as mean (S.E. and are representativeof two individual experiments performed in duplicate.

[0030]FIG. 10A shows the performance of mice with and without treatmenton rotorod latency after closed head injury.

[0031]FIG. 10B shows the weight gain of mice with and without treatmentafter closed head injury.

[0032]FIG. 10C shows the performance of mice with and without treatmentin a water maze latency test after closed head injury.

[0033]FIG. 10D shows the survival of mice with and without treatmentafter closed head injury.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The LRP/α2M receptor. The LRP/α2M receptor is known. In overview,following modification by lipoprotein lipase and the association ofapolipoproteins, very large density lipoproteins (VLDL) and chylomicronbecome remnants, and are cleared hepatically by a receptor-mediatedmechanism. Although recognized as distinct from the low densitylipoprotein (LDL) receptor, the remnant receptor also has a highaffinity for apolipoprotein E, and recognizes the remnant particles viaincorporated apoE moieties. In 1988, this remnant receptor was cloned,and dubbed the LDL receptor-related related protein, or “LRP”. The LRPis a large receptor, with a primary sequence of 4525 amino acids, andbears many structural similarities to other members of the LDL receptorfamily. Like the LDL receptor, the extracellular domain of LRP includesa cysteine-enriched ligand binding domain and EGF precursor homologydomain which are believed to play a role in the acid-dependentdissociation of ligand from the receptor. Unlike the LDL receptor,however, the O-lined sugar domain is not present in the extracellularportion adjacent to the membrane. As with all of the members of the LDLreceptor family, LRP is a transmembrane protein, and is anchored by asingle transmembrane segment. The cytoplasmic tail of the protein is 100amino acids, approximately twice as long as the LDL receptor, andcontains the NP×Y motif, which is believed to be necessary for targetedcoated-pit mediated endocytosis (See, e.g., Krieger M. Herz J.Structures and functions of multiligand lipoprotein receptors:macrophage scavenger receptors and LDL receptor-related protein (LRP).Annual Review of Biochemistry. 63:601-37, 1994. UI: 95069975; Misra U K.Chu C T. Gawdi G. Pizzo S V. The relationship between low densitylipoprotein-related protein/alpha 2-macroglobulin (alpha 2M) receptorsand the newly described alpha 2M signaling receptor. Journal ofBiological Chemistry. 269(28):18303-6, 1994)

[0035] The ApoE genotype in humans has been correlated with outcome in avariety of acute neurological conditions including cerebral hemorrhage,closed head injury, stroke and cognitive deterioration aftercardiopulmonary bypass. See, e.g., Seliger et al., Neurology (Abstract)page A213 (1997); Alberts et al., Stroke 27:183 (abstract)(1996);Connolly et al., Stroke 27:174 (abstract) (1996); Sorbi et al.,Neurology 46:A307 (abstract) (1996); Newman et al., Ann. Thorac. Surg.59:1326 (1995). ApoE is the primarily apolipoprotein produced in thecentral nervous system (CNS) and is upregulated after injury. Laskowitzet al., J. Neuroimmunol. 76:70 (1997).

[0036] ApoE has been demonstrated to have immunomodulatory effects invitro, including suppression of lymphocyte proliferation andimmunoglobulin synthesis after mitogenic challenge. Avila et al., J.Biol. Chem. 257:5900 (1982); Edgington and Curtiss, Cancer Res. 41:3786(1981). ApoE is secreted in large quantities by macrophage afterperipheral nerve injury, and by astrocytes and oligodendrocytes afterCNS injury. Stoll et al., Glia 2:170 (1989); Stoll and Mueller,Neurosci. Lett. 72:233 (1986).

[0037] Apolipoprotein E binds to the low-density lipoprotein (LDL)receptor, as well as to the LDL receptor-related protein (LRP). Theregion of ApoE that is involved in receptor interaction is in thevicinity of amino acid residues 135-160, and is rich in basic aminoacids including arginine and lysine. This interaction of apolipoproteinE and the LDL receptor is important in lipoprotein metabolism. Instudies of the LDL receptor-binding activity of apolipoprotein E, it istypically complexed with phospholipid. The protein has been described asessentially inactive in the lipid-free state. Innerarity et al., J.Biol. Chem. 254:4186-4190 (1979).

[0038] Various amino acid substitutions in the receptor binding regionof ApoE have been studied for their effects on ApoE - LDL receptorbinding. Substitution of either arginine or lysine at residues 136, 142,145 and 146 with neutral residues decreased normal apoE3 bindingactivity. Weisgraber, Advances in Protein Chemistry 45:249 (1994);Lalazar et al., J. Biol. Chem. 263:3542 (1988). No single substitutionof a basic residue within the receptor-binding region of ApoE3completely disrupts LDL receptor binding, suggesting that no one residueis critical for this interaction. It has been postulated that regions ofApoE outside the LDL binding region are necessary to maintain thereceptor-binding region in an active binding conformation. Weisgraber,Advances in Protein Chemistry 45:249 (1994). Dyer et al., J. Biol. Chem.266:15009 (1991), studied lipid-free synthetic peptide fragmentscomprising residues 141-155 of ApoE, and a dimeric peptide of thissequence. No binding activity was observed with the monomer of thispeptide; low levels of binding were observed with the dimer (˜1% of LDLactivity).

[0039] Several receptors that bind ApoE with high affinity have beenidentified, including the scavenger receptor, VLDL receptor, LDLreceptor, and LRP receptors. These three receptors have areas of highsequence similarity. The scavenger receptor is known to be present onmicroglia, and preferentially binds acytylated and oxidized LDL. Thescavenger receptor may be particularly relevant under inflammatory(oxidizing) conditions. Scavenger receptors are also known to beupregulated in microglia after injury. LRP receptors are known to bepresent on macrophages.

[0040] The microglia is the primary immunocompetent cell in the centralnervous system. Acute CNS insult, as well as chronic conditions such asHIV encephalopathy, epilepsy, and Alzheimer's disease (AD) areassociated with microglial activation. McGeer et al., Glia 7:88 (1993);Rothwell and Relton, Cerebrovasc. Brain Metab. Rev. 5:178 (1993);Giulian et al., J. Neuroscience, 16:3139 (1996); Sheng et al., J.Neurochem 63:1872 (1994). Microglial activation results in theproduction of nitric oxide (NO) and other free radical species, and therelease of proteases, inflammatory cytokines (including IL-1β, IL-6 andTNFα), and a neurotoxin that works through the NMDA receptor. Giulian etal., J. Neuroscience, 16:3139 (1996). Microglial activation can beassessed by measuring the production of nitrite, a stable product ofnitric oxide formation. See, e.g., Barger and Harmon, Nature 388:878(1997).

[0041] The present inventors determined that apoE modulates theactivation of glia in the CNS, and further identified a peptide thatsuppresses the activation of microglia. While not wishing to be bound toa single theory, the present inventors hypothesized that ApoE binding toa microglial receptor affects the phenotype of the microglia, decreasingthe responsiveness of the microglia to various activators, and thereforedecreasing the release of inflammatory compounds from the microglia thatwould otherwise occur in the presence of such activators. The ApoE maybe binding to the same receptor as is bound by the activating compounds,or may be binding to a receptor independent from that bound byactivators. In lymphocytes, ApoE has been shown to block activation by avariety of compounds, including LPS, the lectin PHA, and anti-CD3antibody; these activators are known to bind to distinct receptors onlymphocytes. The methods and compounds of the present invention aredesigned to prevent or suppress the receptor-mediated activation ofmicroglia, and thus prevent or reduce the deleterious neurologicaleffects associated with activated microglia. Peptides and othertherapeutic molecules according to the present invention are able tobind to receptors on glia, and decrease the responsiveness of the cellto various activators. In this manner, methods and compounds accordingto the present invention may be used to treat, ameliorate, or preventcertain signs, symptoms, and/or deleterious neurological effects ofacute and/or chronic CNS injury. The effect of the present methods andcompounds may be assessed at the cellular or tissue level (e.g.,histologically or morphometrically), or by assessing a subject'sneurological status. Methods of assessing a subject's neurologicalstatus are known in the art.

[0042] Laskowitz et al., J. Neuroimmunology 76:70 (June 1997) describedexperiments in which mixed neuronal-glial cell cultures fromapoE-deficient mice were stimulated with lipopolysaccharide (LPS). Itwas found that preincubation of the cell cultures with apoE blockedglial secretion of TNFα in a dose-dependent manner. Laskowitz et al., J.Cerebral Blood Flow and Metabolism, 17:753-758 (July 1997) compared theneurologic and histologic outcome of ApoE-deficient mice subjected toocclusion of the cerebral artery for either 60 or 90 minutes, with arecovery period of 24 hours. When subjected to 60 minutes of occlusion,ApoE-deficient mice were reported to have larger infarcts and moresevere hemiparesis than wild-type mice. In mice subjected to 90 minutesof occlusion, mortality was 40% in ApoE-deficient mice compared to 0% inwild-type mice.

[0043] Barger S W and Harmon A D, Nature 388:878 (August 1997) reportedthat treatment of microglia with a secreted derivative of beta-amyloidprecursor protein (sAPP-alpha) activated microglia, induced inflammatoryreactions in microglia, and enhanced the production of neurotoxins bymicroglia. The ability of sAPP-alpha to activate microglia was blockedby prior incubation of the sAPP-alpha protein with apolipoprotein E3 butnot apolipoprotein E4.

[0044] Suitable subjects for carrying out the methods of the presentinvention include male and female mammalian subjects, including humans,non-human primates, and non-primate mammals. Subjects include veterinary(companion animal) subjects, as well as livestock and exotic species.

[0045] The present methods and compounds are useful in preventing,treating, or ameliorating neurological signs and symptoms associatedwith acute CNS injury; as used herein, acute CNS injury includes but isnot limited to stroke (caused by thrombosis, embolism orvasoconstriction), closed head injury, global cerebral ischemia (e.g.,ischemia due to systemic hypotension of any cause, including cardiacinfarction, cardiac arrhythmia, hemorrhagic shock, and post coronaryartery bypass graft brain injury) and intracranial hemorrhage. Further,the present methods and compounds are useful in preventing, treating, orameliorating neurological signs and symptoms associated with chronicneurological disease, including but not limited to Alzheimer's disease(AD) and HIV-associated encephalopathy. The present methods andcompounds are also useful in preventing, treating, or ameliorating theneurological signs and symptoms associated with inflammatory conditionsaffecting the nervous system including the CNS, including but notlimited to multiple sclerosis, vasculitis, acute disseminatedencephalomyelitis, and Guillain-Barre syndrome.

[0046] Stated in a different way, the present methods and compounds areuseful in preventing, suppressing or reducing the activation of glia inthe CNS that occurs as a part of acute or chronic CNS disease. Thesuppression or reduction of glial activation can be assessed by variousmethods as would be apparent to those in the art; one such method is tomeasure the production or presence of compounds that are known to beproduced by activated glia, and compare such measurements to levels ofthe same compounds in control situations. Alternatively, the effects ofthe present methods and compounds in suppressing, reducing or preventingmicroglial activation may be assessed by comparing the signs and/orsymptoms of CNS disease in treated and control subjects, where suchsigns and/or symptoms are associated with or secondary to activation ofmicroglia.

[0047] Ischemic damage to the central nervous system may result fromeither global or focal ischemic conditions. Global ischemia occurs whereblood flow to the entire brain ceases for a period of time, such asduring cardiac arrest. Focal ischemia occurs when a portion of the brainis deprived of normal blood flow, such as during thromboembolyticocclusion of a cerebral vessel, traumatic head injury, edema and braintumors. Much of the CNS damage due to cerebral ischemia occurs duringthe hours or even days following the ischemic condition, and issecondary to the release of cytotoxic products by damaged tissue.

[0048] In Alzheimer's disease, studies indicate that anti-inflammatorydrugs may delay the onset or progression of the disease. Breitner etal., Neurobiol. Aging 16:523 (1995); Rogers et al., Neurology 43:1609(1993). Microglia express markers of activation in AD, suggesting thatcrucial inflammatory events in AD involve microglia. Such activatedmicroglia cluster near amyloid plaques. Griffin et al., J. Neuropath.Exp. Neurol. 54:276 (1995). Microglia are also activated in epilepsy(see Sheng et al., J. Neurochem 63:1872 (1994).

[0049] In subjects with head injuries, AD-like changes are synergisticwith ApoE genotype. The ApoE4 allele has been associated with the extentof amyloid β-protein deposition following head injury. Mayeux et al.,Neurology 45:555 (1995); Nicoll et al., Nat. Med. 1:135 (1995).

[0050] As used herein, the terms “combating”, “treating” and“ameliorating” are not necessarily meant to indicate a reversal orcessation of the disease process underlying the CNS condition afflictingthe subject being treated. Such terms indicate that the deleterioussigns and/or symptoms associated with the condition being treated arelessened or reduced, or the rate of progression is reduced, compared tothat which would occur in the absence of treatment. A change in adisease sign or symptom may be assessed at the level of the subject(e.g., the function or condition of the subject is assessed), or at atissue or cellular level (e.g., the production of markers of glialactivation is lessened or reduced). Where the methods of the presentinvention are used to treat chronic CNS conditions (such as Alzheimer'sdisease), the methods may slow or delay the onset of symptoms such asdementia, while not necessarily affecting or reversing the underlyingdisease process.

[0051] Active Compounds. Active compounds that may be used to carry outthe present invention include ligands or agonists that specificallyand/or selectively bind to the LRP/α2M receptor. Examples of suchcompounds include, but are not limited to, 1) alpha 2 macroglobulin; 2)pseudomonas exotoxin; 3) lipoprotein lipase; 4) apolipoprotein E; 5)oxidized and/or acetylated LDL; 6) receptor associated protein (RAP); 7)remnant particles; 8) low density lipoprotein (LDL); 9) high denitylipoprotein (HDL); 10) lactoferrin; 11) tissue plasminogen activator(tPA); 12) urine plasminogen activator (uPA); etc.

[0052] Amino acid residues 100-200 of each isoform of the ApoE moleculecomprise the ApoE receptor binding region. More specifically, thereceptor binding region of ApoE is within amino acid residues 130-160 ofeach isoform of the ApoE molecule (SEQ ID NO:4 and SEQ ID NO:5), andmore specifically is within amino acid residues 140-155 (HLRKLRKRLLRDADDL) (SEQ ID NO:1). See, e.g., Weisgraber, Apolipoprotein E:Structure-Function Relationships, Advances in Protein Chemistry 45:249(1994). The amino acid interchanges that define the E2, E3 and E4isoforms are not found within the region of amino acid residues 140-155,but do influence the overall structure of the apolipoprotein molecule.ApoE2 and ApoE3 molecules form covalently bound homodimers; ApoE4molecules do not.

[0053] As used herein, the term homodimer refers to a molecule composedof two molecules of the same chemical composition; the term heterodimerrefers to a molecule composed of two molecules of differing chemicalcomposition.

[0054] The present inventors utilized a 9-mer monomer having an aminoacid sequence LRKLRKRLL (SEQ ID NO:2). This 9 amino acid sequence isfound within the larger ApoE receptor binding sequence region identifiedabove, and is found at amino acid positions 141-149 of ApoE. The presentinventors constructed a dimer of SEQ ID NO:2, i.e., a peptide having anamino acid sequence of LRKLRKRLL LRKLRKRLL (SEQ ID NO:3). Peptides ofSEQ ID NO:3 suppressed microglial activation in a dose-dependentfashion. Use of the monomer (monomer peptides of SEQ ID NO:2) did notsuppress microglial activation. (See FIG. 2).

[0055] The present inventors further utilized a 20-mer monomer having anamino acid sequence TEELRVRLAS HLRKLRKRLL (SEQ ID NO:6). This 20 aminoacid sequence is found at amino acid positions 130-149 of ApoE, andcomprises the 9-mer SEQ ID NO:2. Peptides of SEQ ID NO:6 suppressedmicroglial activation in a dose-dependent fashion (see FIGS. 4-7).

[0056] Clay et al., Biochemistry 34:11142 (1995) reported that dimericpeptides of amino acids 141-155 or 141-149 were both cytostatic andcytotoxic to T lymphocytes in culture. Cardin et al. Biochem BiophysRes. Commun. 154:741 (1988) reported that a peptide of apoE 141-155inhibited the proliferation of lymphocytes. A peptide consisting of atandem repeat of amino acids 141-155, as well as longer monomericpeptides comprising the 141-155 region, was found to cause extensive andspecific degeneration of neurites from embryonic chicks in vitro.Crutcher et al., Exp. Neurol. 130:120 (1994). These authors suggestedthat peptide sequences associated with apoE might contribute directly toneurodegenerative processes.

[0057] Peptides of the present invention may be produced by standardtechniques as are known in the art.

[0058] Active compounds (or “active agents”) useful in the methods ofthe present invention include those that compete with a peptide of SEQID NO:3, and/or a peptide of SEQ ID NO:6 in binding to microglialreceptors to thereby prevent or suppress activation of the microglia bymolecules that would otherwise activate microglia.

[0059] Peptides useful in the present methods include those comprisingthe ApoE LDL receptor binding sequence (including multiple repeatsthereof, including but not limited to dimers and trimers); andconjugates of two or more peptides, each of which comprises a peptide asdescribed herein or a peptide comprising the LDL receptor bindingsequence. One ApoE receptor binding sequence is provided in SEQ ID NO:1.A preferred peptide comprises or consists of multiple repeats of SEQ IDNO:2, preferably dimers thereof. Thus, a preferred peptide useful in thepresent methods is SEQ ID NO:3 (a tandem repeat of LRKLRKRLL), orpeptides comprising SEQ ID NO:3. Further preferred peptides comprise orconsist of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0060] The ability of a linear tandem repeat of amino acids 141-155 (the141-155 dimer) to bind the LDL receptor was studied by Dyer et al., J.Lipid Research 36:80 (1995). A series of modified peptides wasconstructed and assessed for LDL binding ability. These authors reportthat deletion of the charged amino terminal residues (including arg142and lys143) in 145-155 or 144-150 dimers abolished the LDL receptoractivities of the peptides. These authors conclude that LDL-receptorbinding activity of the 141-155 dimer is dependent on at least twoclusters of basic amino acids present on the hydrophilic face of theamphipathic alpha-helix of the 141-155, 141-150, 141-155 (lys143→ala)and 141-155 (arg150→ala) dimer peptides. Dyer et al., J. Biol. Chem.266:15009 (1991) reported that a self-conjugate of peptide 141-155, anda peptide consisting of a tandem repeat of 141-155, were able to inhibitboth lymphocyte proliferation and ovarian androgen production. Dyer etal., J. Biol. Chem. 266:22803 (1991) investigated the LDL bindingability of a dimeric 141-155 tandem peptide, and a trimeric 141-155peptide. Binding was decreased with amino acid substitutions ofLys-143→Ala, Leu144→Pro, and Arg150→Ala.

[0061] Lalazar et al., J. Biol. Chem. 263:3542 (1988) investigatedvariants of ApoE for binding to the LDL receptor. When neutral aminoacids were substituted for basic residues at positions 136, 140, 143,and 150, binding activity was reduced. Where proline was substituted forleucine144 or alanine152, binding was reduced. However, slightlyenhanced receptor binding was displayed by a variant in which argininewas substituted for serinel39 and alanine was substituted for leucine149.

[0062] Compounds that are useful in the present method include thosewhich act as antagonists for the microglial receptor bound by peptidesof SEQ ID NO:3 and/or SEQ ID NO:6. Antibodies that selectively targetand bind to this receptor can also be used as antagonists of microglialactivation according to the present invention. Such antibodiesselectively or specifically bind to the receptor bound by peptides ofSEQ ID NO:3 and/or peptides of SEQ ID NO:6.

[0063] Peptides of SEQ ID NO:3, SEQ ID NO:6, or conformational analoguesthereof, are an aspect of the present invention. Such compounds arepeptides or peptidomimetics having a core sequence of amino acids with aconformation in aqueous solution that interacts with receptor moleculeson glial cells to block the activation of glial cells that wouldotherwise occur in conjunction with acute or chronic CNS injury, orexposure to known activators of microglia such as LPS. Stated anotherway, such compounds are characterized by the ability to compete withpeptides of SEQ ID NO:3 and/or peptides of SEQ ID NO:6 for binding tomicroglia, and by their ability to suppress microglial activation byknown activators such as LPS.

[0064] Another variation of the therapeutic peptides of the presentinvention is the linking of from one to five amino acids or analogues tothe N-terminal or C-terminal amino acid of the therapeutic peptide.Analogs of the peptides of the present invention may also be prepared byadding from one to five additional amino acids to the N-terminal,C-terminal, or both N- and C-terminals, of an active peptide, where suchamino acid additions do not adversely affect the ability of the peptideto bind to microglia at the site bound by a peptide of SEQ ID NO:3and/or SEQ ID NO:6.

[0065] Changes in the amino acid sequence of peptides can be guided byknown similarities among amino acids and other molecules or substituentsin physical features such as charge density, hydrophobicity,hydrophilicity, size and configuration, etc. For example, the amino acidThr may be replaced by Ser and vice versa, and Leu may be replaced byIle and vice versa. Further, the selection of analogs may be made bymass screening techniques known to those skilled in the art (e.g.,screening for compounds which bind to microglia at the receptor bound bya peptide of SEQ ID NO:3 and/or SEQ ID NO:6). A preferred exchange is toreplace Ser with Arg, to increase the arginine content of the peptide;examples include peptides of or comprising SEQ ID NO:7, SEQ ID NO:8 orSEQ ID NO:9. A further preferred exchange is to substitute alanine forleucine149.

[0066] Peptides of the present invention may also be characterized asshort peptides of from about 20 amino acids, 22 amino acids, 24 aminoacids, 26 amino acids, 28 amino acids, 30 amino acids, 35 amino acids,or 40 amino acids, up to about 22 amino acids, 24 amino acids, 26 aminoacids, 28 amino acids, 30 amino acids, 35 amino acids, 40 amino acids,45 amino acids, 50 amino acids or more, where the peptides comprise the18-amino acid sequence LRKLRKRLL LRKLRKRLL (SEQ ID NO:3), or variantsthereof that retain the receptor binding ability of peptides of SEQ IDNO:3. A preferred peptide useful in the present invention is oneconsisting of or comprising SEQ ID NO:3. Where longer peptides areemployed, those incorporating amino acid sequences derived from the ApoEsequence immediately surrounding amino acid residues 141-149 arepreferred. Where peptides longer than 18 amino acids are employed, it iscontemplated that they may include virtually any other amino acidsequences so long as the resultant peptide maintains its ability to bindto microglial and suppress microglia activation in acute and chronic CNSinflammation. The present invention includes those variations of theApoE sequence at 141-149 which are known to retain the ability LDLreceptor- binding ability. Synthetic peptides may further be employed,for example, using one or more D-amino acids in place of L-amino acids,or by adding groups to the N- or C-termini, such as by acylation oramination.

[0067] Peptides of the present invention may also be characterized asshort peptides of from about 10 amino acids, 12 amino acids, 14 aminoacids, 15 amino acids, 18 amino acids, 20 amino acids, 22 amino acids,24 amino acids, 26 amino acids, 28 amino acids, 30 amino acids, 35 aminoacids, or 40 amino acids, up to about 15 amino acids, 22 amino acids, 24amino acids, 26 amino acids, 28 amino acids, 30 amino acids, 35 aminoacids, 40 amino acids, 45 amino acids, 50 amino acids or more, where thepeptides comprise the 9-amino acid sequence LRKLRKRLL (SEQ ID NO:2), orvariants thereof that retain the receptor binding ability of peptides ofSEQ ID NO:3 and/or SEQ ID NO:6. A preferred peptide useful in thepresent invention is one consisting of or comprising the apoE receptorbinding region; a particularly preferred peptide consists of orcomprises SEQ ID NO:6. Where longer peptides are employed, thoseincorporating amino acid sequences derived from within the apoE receptorbinding region, or the ApoE sequence immediately surrounding the apoEreceptor binding region, are preferred, although it is contemplated thatthese peptides may include virtually any other amino acid sequences solong as the resultant peptide maintains its ability to bind to microgliaand suppress microglia activation in acute and chronic CNS inflammation.The present invention includes those variations of the ApoE sequence at141-149 which are known to retain the ability LDL receptor-bindingability. Synthetic peptides may further be employed, for example, usingone or more D-amino acids in place of L-amino acids, or by adding groupsto the N- or C-termini, such as by acylation or amination.

[0068] The peptides of the present invention include not only naturalamino acid sequences, but also peptides which are analogs, chemicalderivatives, or salts thereof. The term “analog” or “conservativevariation” refers to any polypeptide having a substantially identicalamino acid sequence to the therapeutic peptides identified herein, andin which one or more amino acids have been substituted with chemicallysimilar amino acids. For example, a polar amino acid such as glycine orserine may be substituted for another polar amino acid; a basic aminoacid may be substituted for another basic amino acid, or an acidic aminoacid may be substituted for another acidic amino acid; or a non-polaramino acid may be substituted for another non-polar amino acid. Thereterm “analog” or “conservative variation” as used herein also refers toa peptide which has had one or more amino acids deleted or added to apolypeptide of the present invention, but which retains a substantialsequence similarity (at least about 85% sequence similarity, andpreferably at least 90%, 92%, 94%, 95%, 96%, 98% or even 99% sequencesimilarity), where the peptide retains the ability to suppressmicroglial activation as described herein.

[0069] The amino acids constituting peptides of the present inventionmay be of either the L-configuration or the D-configuration. Therapeuticpeptides of the present invention may be in free form or the form of asalt, where the salt is pharmaceutically acceptable.

[0070] As used herein, the term “administering to the brain of asubject” refers to the use of routes of administration, as are known inthe art, that provide the compound to the central nervous systemtissues, and in particular the brain, of a subject being treated.

[0071] Preferably, the compounds of the present invention are used incombination with a pharmaceutically acceptable carrier. The presentinvention thus also provides pharmaceutical compositions suitable foradministration to mammalian subjects. Such compositions comprise aneffective amount of the compound of the present invention in combinationwith a pharmaceutically acceptable carrier. The carrier may be a liquid,so that the composition is adapted for parenteral administration, or maybe solid, i.e., a tablet or pill formulated for oral administration.Further, the carrier may be in the form of a nebulizable liquid or solidso that the composition is adapted for inhalation. When administeredparenterally, the composition should by pyrogen free and in anacceptable parenteral carrier. Active compounds may alternatively beformulated encapsulated in liposomes, using known methods. Additionally,the intranasal administration of peptides to treat CNS conditions isknown in the art (see, e.g., U.S. Pat. No. 5,567,682 to Pert, regardingintranasal administration of peptide T to treat AD). (All patentsreferenced herein are intended to be incorporated by reference herein intheir entirety.) Preparation of a compound of the present invention forintranasal administration may be carried out using techniques as areknown in the art.

[0072] Pharmaceutical preparations of the compounds of the presentinvention may optionally include a pharmaceutically acceptable diluentor excipient.

[0073] An effective amount of the compound of the present invention isthat amount that decreases microglial activation compared to that whichwould occur in the absence of the compound; in other words, an amountthat decreases the production of neurotoxic compounds by the microglia,compared to that which would occur in the absence of the compound. Theeffective amount (and the manner of administration) will be determinedon an individual basis and will be based on the specific therapeuticmolecule being used and a consideration of the subject (size, age,general health), the condition being treated (AD, acute head injury,cerebral inflammation, etc.), the severity of the symptoms to betreated, the result sought, the specific carrier or pharmaceuticalformulation being used, the route of administration, and other factorsas would be apparent to those skilled in the art. The effective amountcan be determined by one of ordinary skill in the art using techniquesas are known in the art. Therapeutically effective amounts of thecompounds described herein may be determined using in vitro tests,animal models or other dose-response studies, as are known in the art.

[0074] The compounds of the present invention may be administeredacutely (i.e., during the onset or shortly after events leading tocerebral inflammation or ischemia), or may be administeredprophylactically (e.g., before scheduled surgery, or before theappearance of neurologic signs or symptoms), or administered during thecourse of a degenerative disease to reduce or ameliorate the progressionof symptoms that would otherwise occur. The timing and interval ofadministration is varied according to the subject's symptoms, and may beadministered at an interval of several hours to several days, over atime course of hours, days, weeks or longer, as would be determined byone skilled in the art.

[0075] The typical daily regime may be from about 0.01 μg/kg body weightper day, from about 10 μg/kg body weight per day, from about 100 μg/kgbody weight per day, from about 1000 μg/kg body weight per day, fromabout 10,000 μg/kg body weight per day, from about 100,000 μg/kg bodyweight per day.

[0076] The blood-brain barrier presents a barrier to the passivediffusion of substances from the bloodstream into various regions of theCNS. However, active transport of certain agents is known to occur ineither direction across the blood-brain barrier. Substances that mayhave limited access to the brain from the bloodstream can be injecteddirectly into the cerebrospinal fluid. Cerebral ischemia andinflammation are also known to modify the blood-brain barrier and resultin increased access to substances in the bloodstream.

[0077] Administration of a compound directly to the brain is known inthe art. Intrathecal injection administers agents directly to the brainventricles and the spinal fluid. Surgically-implantable infusion pumpsare available to provide sustained administration of agents directlyinto the spinal fluid. Lumbar puncture with injection of apharmaceutical compound into the cerebrospinal fluid (“spinalinjection”) is known in the art, and is suited for administration of thepresent compounds.

[0078] Pharmacologic-based procedures are also known in the art forcircumventing the blood brain barrier, including the conversion ofhydrophilic compounds into lipid-soluble drugs. The active agent may beencapsulated in a lipid vesicle or liposome.

[0079] The intra-arterial infusion of hypertonic substances totransiently open the blood-brain barrier and allow passage ofhydrophilic drugs into the brain is also known in the art. U.S. Pat. No.5,686,416 to Kozarich et al. discloses the co-administration of receptormediated permeabilizer (RMP) peptides with compounds to be delivered tothe interstitial fluid compartment of the brain, to cause an increase inthe permeability of the blood-brain barrier and effect increaseddelivery of the compounds to the brain. Intravenous or intraperitonealadministration may also be used to administer the compounds of thepresent invention.

[0080] One method of transporting an active agent across the blood-brainbarrier is to couple or conjugate the active agent to a second molecule(a “carrier”), which is a peptide or non-proteinaceous moiety selectedfor its ability to penetrate the blood-brain barrier and transport theactive agent across the blood-brain barrier. Examples of suitablecarriers include pyridinium, fatty acids, inositol, cholesterol, andglucose derivatives. The carrier may be a compound which enters thebrain through a specific transport system in brain endothelial cells.Chimeric peptides adapted for delivering neuropharmaceutical agents intothe brain by receptor-mediated transcytosis through the blood-brainbarrier are disclosed in U.S. Pat. No. 4,902,505 to Pardridge et al.These chimeric peptides comprise a pharmaceutical agent conjugated witha transportable peptide capable of crossing the blood-brain barrier bytranscytosis. Specific transportable peptides disclosed by Pardridge etal. include histone, insulin, transferrin, and others. Conjugates of acompound with a carrier molecule, to cross the blood-brain barrier, arealso disclosed in U.S. Pat. No. 5,604,198 to Poduslo et al. Specificcarrier molecules disclosed include hemoglobin, lysozyme, cytochrome c,ceruloplasmin, calmodulin, ubiquitin and substance P. See also U.S. Pat.No. 5,017,566 to Bodor.

[0081] An alternative method of administering peptides of the presentinvention is carried out by administering to the subject a vectorcarrying a nucleic acid sequence encoding the peptide, where the vectoris capable of entering brain cells so that the peptide is expressed andsecreted, and is thus available to microglial cells. Suitable vectorsare typically viral vectors, including DNA viruses, RNA viruses, andretroviruses. Techniques for utilizing vector deliver systems andcarrying out gene therapy are known in the art. Herpesvirus vectors area particular type of vector that may be employed in administeringcompounds of the present invention.

[0082] Screening Methods. Also disclosed herein are methods of screeningcompounds for the ability to prevent or reduce microglial activationunder conditions of cerebral ischemia or cerebral inflammation. Suchmethods comprise contacting an activated microglial cell with a testcompound, and detecting whether the test compound binds to microglia atthe same receptor at which peptides of SEQ ID NO:3 and/or SEQ ID NO:6bind. The contacting step may be carried out in vitro, for example incell culture. A competitive binding assay may be used to detect whetherthe test compound binds to the same receptor that is bound by peptidesof SEQ ID NO:3 and/or SEQ ID NO:6.

[0083] An additional method of screening a test compound for the abilityto suppress microglial activation comprises incubating an activatedmicroglial cell culture with a test compound, and measuring at least onemarker of microglial activation. A decrease in a marker of microglialactivation (compared to the level of that marker that would occur in theabsence of the test compound) indicates that the test compound is ableto suppress, prevent or reduce microglial activation. An exemplarymarker of microglial activation is the production of nitric oxide.

[0084] A further method of screening a test compound for the ability tosuppress microglial activation involves pre-incubating a microglial cellculture with a test compound, then incubating the microglial cellculture with a compound that is known to activate microglia. At leastone marker of microglial activation is then measured, and a decrease inthe activation marker (compared to that which occurs in the absence ofthe pre-incubation step) indicates that the test compound is able toaffect microglial activation. An exemplary marker of microglialactivation is the production of nitric oxide.

[0085] Atherosclerosis. It known that the inflammatory process mediatesan aspect of the atherosclerotic process. See, e.g., Hansson, Basic Res.Cardiol., 89(1):41 (1994); Berliner et al., Circulation 91:2488 (1995);Watanabe et al., Int. J. Cardiol. 54:551 (1997). ApoE is known to besecreted by macrophages locally at blood vessel walls (although theamount secreted by macrophages in an individual is trivial compared tothe amount of ApoE produced by the liver). In the classic model ofatherosclerosis, ApoE functions to remove cholesterol from the bloodstream and deliver it to macrophages or to the liver. However, it hasbecome apparent that ApoE secreted by macrophages at the blood vesselwall decreases atherosclerotic plaque formation, independent of anylipid metabolism effects. ApoE-deficient mice are accepted as a model ofhypercholesteremia and atherosclerotic disease; providing ApoE-secretingmacrophages to such mice dramatically decreases atherosclerotic plaqueformation. Linton et al., Science, 267:1034 (1995). Conversely,replacing a wild-type mouse's macrophages with ApoE-deficientmacrophages accelerates atherosclerotic changes, even though the animalcontinues to produce ApoE by the liver. Fazio et al., Proc. Natl. Acad.Sci. 94:4647 (1997). In atherosclerosis it is hypothesized that ApoE,via a receptor-mediated event, downregulates macrophage activation inthe vicinity of blood vessel walls. Such down-regulation of macrophageactivation interrupts or interfers with the cascade of events associatedwith atherosclerotic plaque formation, to thereby reduce or slow theformation of atherosclerotic lesions. The cascade of events known to beassociated with atherosclerosis includes smooth muscle cell andendothelial cell proliferation, and foam cell formation; evidence existsthat ApoE downregulates each of these processes. ApoE thus affects thepresence and progression of atherosclerosis in vivo, independent of itseffects on lipids. The progression of atherosclerosis may be assessed bymeasuring the amount or size of atherosclerotic plaques, or thepercentage of the blood vessel blocked by an atherosclerotic lesion, orthe rate of growth of such plaques.

[0086] The present inventors have for the first time demonstrated thatApoE transduces a calcium-mediated signal (Ca²⁺/inositol triphosphatesignal transduction) in macrophage, indicating that ApoE modifiesmacrophage function by downregulating macrophage activation and,therefore, subsequent inflammation. Peptides, compounds, methods andpharmaceutical formulations as described herein in relation to microgliaand CNS disease are accordingly useful in methods of suppressing theactivation of macrophages to suppress, prevent, or slow atherosclerosis.Atherosclerosis refers to the thickening of the arterial intima andaccumulation of lipid in artherosclerotic plaques. Administration ofcompounds of the present invention to treat or prevent atherosclerosismay be by any means discussed herein as well as other suitable methodsthat are known in the art. When using the present compounds to prevent,slow or treat atherosclerotic changes, it is apparent that they need notbe formulated to pass through the blood brain barrier. Conditions thatmay be treated by the present method include atherosclerosis of thecoronary arteries; arteries supplying the Central Nervous system, suchas carotid arteries; arteries of the peripheral circulation or thesplanchnic circulation; and renal artery disease. Administration, suchas parenteral administration, may be site-specific or into the generalblood stream.

[0087] The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 Microgial Nitric Oxide Production: Materials and Methods

[0088] This study examined the role of endogenous apoE in modulatingmicroglial nitric oxide (NO) production, as measured by nitriteaccumulation following lipopolysaccharide (LPS) stimulation ofmicroglia.

[0089] Culture preparation and characterization: Mixed glial cellcultures were prepared from: (a) wildtype (C57/B16; JacksonLaboratories) mouse pups; (b) ApoE deficient mutant mouse pups(ApoE-deficient mice), and (c) transgenic mouse pups expressing humanApoE3 but not murine ApoE (ApoE3 mice). See Xu et al., Neurobiol. Dis.3:229 (1996) regarding the creation and characterization of thetransgenic mice. Mixed glial cell cultures were prepared as has beendescribed. See McMillian et al., Neurochem. 58:1308 (1992); Laskowitz etal., J. Neuroimmunol. 76:70 (1997). Briefly, brains were removed from2-4 day old pups, cleaned of membranes and blood vessels, mechanicallydispersed in Ca⁺²-free media, and collected by centrifugation. Cellswere then plated in DMEM/F12 (containing 10% fetal calf serum, 1%penicillin/streptomycin, Gibco #15070), one brain per 25 cm flask. Mixedneuronal/glial preparations were grown in humidified incubators untilconfluent (3-5 weeks).

[0090] The percentage of microglia, astrocytes and neurons werequantified to demonstrate that cultures prepared from ApoE-deficient andApoE3 mice had comparable glial populations. Immunostaining wasperformed using antibodies to glial fibrillary acidic protein (GFAP;SIGMA®; 1:500 dilution) and tau protein (SIGMA®; 1:500 dilution) toestimate numbers of astrocytes and neurons, and peroxidase-coupledBandeiraea simplifolica B4 isolectin and naphthyl acetate esterasestaining was used to detect microglia. Laskowitz et al., J.Neuroimmunol. 76:70 (1997). A mixed neuronal-glial culture system wasused, as this most closely approximates the normal CNS milieu, andallows glia-glia interactions, which play a role in the inflammatorycascade.

[0091] Comparable glial populations were confirmed usingsemi-quantitative Western blot analysis performed for astrocytes (αGFAP;SIGMA®), neurons (αtau; SIGMA®) and microglia (Bandeiraea simplifolicaB4 isolectin; SIGMA®). Cellular protein was harvested at the end ofexperiments and 50 μg protein from each sample was separated bypolyacrilamide gel electrophoresis and the protein was transferred tonylon membranes. Non-specific binding of antisera and lectin was blockedby preincubation of the membrane in 4% dried milk, 0.1% Triton X-100.Membranes were incubated overnight with antibodies or 1 μg/ml B4isolectin. After extensive washing in phosphate-buffered saline, boundantibody or lectin was visualized by an ABC kit (Vector, Burlingame,Calif.), using diaminobenzidine as substrate.

[0092] Culture Stimulation: Cultures were plated in serum-free mediaafter washing cells once with this media, and stimulated with LPS 100ng/ml (SIGMA®). Aliquots were taken at 24 and 60 hours for nitriteassay.

[0093] Nitrite Quantification: The production of NO was assessed bymeasuring the accumulation of nitrite, which was quantified using acolorimetric reaction with Griess reagent (0.1%N-1-naphthylethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5%H₃PO₄). Absorbance was measured at 570 nm by spectrophotometry. Thesensitivity of this assay is approximately 0.5 μM.

[0094] Statistical Analysis: Data were compared by ANOVA and the FischerLSD multiple range test; p<0.05 was considered significant.

EXAMPLE 2 Microglial Nitric Oxide Production: Results

[0095] Culture Characterization: No significant differences were foundin glial populations among the cultures prepared from ApoE-deficient,ApoE3, and wild-type mice. Cultures comprised approximately 70%astrocytes, 15% microglia and 15% neurons. Comparisons of cellularpreparations from wildtype mice, ApoE-deficient mice and ApoE3 miceshowed no differences in glial populations. In particular, levels ofmicroglia (the primary effector cells for NO production) were comparablein all three culture preparations, as detected by lectin binding (datanot shown).

[0096] ApoE-deficient mouse cultures showed robust nitrite responsesduring the first 24 hours of exposure to LPS. This enhanced response was6-fold greater than that observed with microglia from control animals(p=0.0001; FIG. 1). Cultures from transgenic mice in which murine apoEis replaced with human ApoE3 show weak responses to LPS that were notsignificantly different than responses of wildtype animals (p=0.64 andp=0.2 at 24 and 60 hours, respectively). By 60 hours, increased nitriteaccumulation was observed in response to LPS in wildtype and ApoE3transgenic mouse preparations, although there was still a significantlygreater amount of nitrite in the apoE deficient culture as compared tocontrols (p=0.04%; FIG. 1).

[0097] The above studies show that ApoE deficient mixed neuronal-glialcultures respond differently to LPS stimulation than glial culturesprepared from mice expressing native murine ApoE3 or those expressingthe human ApoE3 isoform. These results are consistent with ApoE being abiologically relevant mediator of the CNS response to injury. Thesestudies demonstrate that endogenous ApoE modulates glial secretion ofLPS-stimulated nitric oxide production, and suggest that one function ofendogenous ApoE produced within the brain is to suppress microglialreactivity and thus alter the CNS response to acute and chronic injury.

EXAMPLE 3 Suppression of Microglial Activation by Peptides of SEQ IDNO:3

[0098] Enriched microglia primary cultures were prepared from the brainsof apoE deficient mouse pups as described in Example 1, above. Themicroglia were stimulated with lipopolysaccharide (100 ng/ml) toactivate the microglia as described in Example 1. Activated microgliasecrete inflammatory cytokines and nitric oxide; the secretion of nitricoxide was used in the present experiment as a marker of microglialactivation. Nitric oxide production was assessed as described in Example1.

[0099] Peptides of SEQ ID NO:3 were added to cultures of activatedmicroglia, in dosages of from 0 μM to 1000 μM. A dose-dependent decreasein nitric oxide secretion was observed after 48 hours (FIG. 2). Theadministration of a peptide of SEQ ID NO:2 in a dose of 2 mM did notresult in any apparent decrease in nitric oxide secretion (FIG. 2). Themonomer peptide of SEQ ID NO:2 acted as a control to establish that theobserved results are not due to any non-specific peptide effect.

EXAMPLE 4 Effect of ApoE on Macrophage

[0100] Intracellular signaling pathways of ApoE were investigated usingperitoneal macrophage.

[0101] Thioglycolate-elicited peritoneal macrophage were harvested from8-week old C57-BL6 mice, and plated at a density of 4×10⁵ cells on glasscoverslips, loaded with 2.5 μM Fura-2/AM for thirty minutes, and washedwith Hanks buffered solution containing 75 μM calcium. After exposure to5 nM human recombinant apoE3 or E4, intracellular calcium was measuredby Zeiss digital microscopy. As shown in FIG. 3A, ApoE causedintracellular mobilization of intracellular calcium in the macrophage.Preincubation with 100 molar excess of Receptor Associated Protein (RAP)did not block this effect; RAP is a physiological antagonist to LRP andblocks the function of LRP.

[0102] Macrophage were also plated at a density of 2×10⁶ cells/well,labeled with ³H-myoinositol (8 μC/ml) 16 hours at 37 degrees, andexposed to human ApoE3 or ApoE4 (5 nM). Control cells were exposed tovehicle but not ApoE. Results are shown in FIG. 3B; values are expressedas the percent change in inositol trisphosphate in treated cells ascompared to control cells.

[0103] Exposure of peritoneal macrophage to ApoE induced a rise inintracellular calcium associated with turnover of inositoltris-phosphate (FIGS. 3A and 3B). The present results indicate that ApoEinitiates a signal transduction pathway that affects and modifiesmacrophage function. The present data suggest that ApoE downregulatesmacrophage activation and inflammation; macrophage activation andinflammation is known to contribute to the atherosclerotic process.

EXAMPLE 5 Suppression of Microglial Activation Using Peptides of SEQ IDNO:6

[0104] A 20-amino acid peptide derived from the receptor binding regionof apoE, containing amino acids 130-149 (SEQ ID NO:6) was preparedaccording to methods known in the art.

[0105] Primary murine microglial cultures were prepared as described inExample 1, from apoE deficient mouse pups. In some cultures themicroglia were activated with lipopolysaccharide (100 ng/ml), asdescribed in Example 1.

[0106] Peptides of SEQ ID NO:6 were added to cultures of activated andnon-activated microglia, in dosages of 0 μM (control), 10 μM, 100 μM and1000 μM (FIG. 4). Each dosage level of peptide was tested alone(squares) and in combination with LPS (100 ng/ml; circles). Theproduction of TNFα was then measured 24 hours after addition of thepeptides. A decrease in TNFα production by activated microglia (comparedto control culture) was observed with each peptide dose used (FIG. 4,circles). Data in FIG. 4 is presented in at least triplicate at eachdose; error bars represent standard error of the mean).

[0107] These results indicate that peptides of SEQ ID NO:6 suppresscytokine release from activated glial cells.

EXAMPLE 6 Cytotoxicity of Peptides of SEQ ID NO:6

[0108] The toxic effects of peptides of SEQ ID NO:6 was investigated.Cultures of activated (LPS) and non-activated microglia, as described inExample 5, were used. Peptides having SEQ ID NO:6 were added to cellcultures in amounts of 0 μM (control), 10 μM, 100 μM and 1000 μM; eachdosage level of peptide was tested alone (squares) and in combinationwith LPS (100 ng/ml; circles). Cell viability was then measured byoptical density 24 hours after addition of the peptides.

[0109] As shown in FIG. 5, optical density was approximately the same incultures receiving 0 μM and 10 μM of peptide, but decreased in culturesreceiving 100 μM or 1000 μM. These results, taken with the results ofExample 5, indicate that a non-toxic concentration of a peptide of SEQID NO:6 is sufficient to suppress glial cytokine release.

EXAMPLE 7 Suppression of Glial Cytokines and Cytotoxicity of Peptides ofSEQ ID NO:6

[0110] The experiments as described in Examples 5 and 6 were repeatedusing a peptide doses of 0 μM (control), 1 μM, 10 μM, 100 μM and 1000μM. Each dosage level of peptide was tested alone (squares) and incombination with LPS (100 ng/ml; circles). The production of TNFα wasmeasured 24 hours after administration of the peptides, and results areshown in FIG. 6. The optical density of the cell cultures was alsomeasured (at 24 hours) to assess cell viability; results are shown inFIG. 7.

[0111] These results show that microglial cytokine release wassuppressed in cell cultures receiving as little as 1 μM of peptide, butcytotoxic effects were seen only in cultures receiving much larger dosesof peptide. The results of examples 5-7 indicate that non-toxicconcentrations of peptides comprising the receptor binding region ofapoE are able to suppress cytokine release from activated microglia.

EXAMPLE 8 In vivo Treatment of Focal Ischemia

[0112] A murine model of focal ischemia-reperfusion is used to assessthe effects of intrathecal, intravenous or intraperitonealadministration of small therapeutic peptides (fewer than 30 amino acidsin length) comprising the apoE LDL receptor region. One such peptide hasSEQ ID NO:6.

[0113] Wild-type mice are subjected to middle cerebral artery occlusionand reperfusion according to techniques known in the art (see, e.g.,Laskowitz et al., J. Cereb. Blood Flow Metab. 17:753 (July 1997)). Onegroup of mice (wild-type control) receives no treatment after cerebralartery occlusion; in a similar group (wild-type treatment group) eachmouse receives intrathecal, intraperitoneal or intravenous injection ofa therapeutic peptide. Therapeutic peptides may be injected in varyingdoses, using the in vitro data provided above as an initial guide.

[0114] Each animal is evaluated neurologically at a predetermined timeafter reperfusion (e.g., 24 hours after reperfusion) (see, e.g.Laskowitz et al., J. Cereb. Blood Flow Metab. 17:753 (July 1997)). Afterneurological examination each mouse is anesthetized and sacrificed andthe brain is sectioned and stained, and infarct volume is measured.Neurological outcome and infarct size is compared between control andtreatment groups.

[0115] The above experiments may be repeated using apoE deficient mice.

EXAMPLE 9 In vivo Treatment of Global Ischemia

[0116] A murine model of global ischemia, adapted from the rat twovessel occlusion model of global ischemia, is used to assess the effectsof intrathecal administration of small therapeutic peptides (fewer than30 amino acids in length) comprising the apoE LDL receptor region. Onesuch peptide has SEQ ID NO:6.

[0117] Wild-type mice (21±1 grams) are fasted overnight, anesthetizedwith halothane or another suitable anesthetic, intubated andmechanically ventilated. The right internal jugular vein and femoralartery are cannulated. Pericranial temperature is held at 37.0 C. Thecarotid arteries are occluded and mean arterial pressue is reduced to 35mmHg with 0.3 mg intra-arterial trimethaphan and venous exsanguination.Ten minutes later ischemia is reversed. Control mice receive noadditional treatment, test mice receive intrathecal, intravenous orintraperitoneal injection of a therapeutic peptide. Peptides may beinjected at varying doses, using the in vitro data provided herein as aguide.

[0118] Each animal is evaluated neurologically at a predetermined time(e.g., 1, 3 or 5 days after reperfusion), using known neurologicaltesting procedures (see, e.g., Laskowitz et al., J. Cereb. Blood FlowMetab. 17:753 (July 1997)). After neurological evaluation, each animalis anesthetized and sacrificed and the brain injury is assessed usingmethods known in the art. For example, brains may be perfusion fixed insitu, then sectioned, stained and examined by light microscopy, forexample, to determine injury to the CA1 sector of the hippocampus, andviable and non-viable neurons counted and compared.

[0119] Neurological outcome and brain injury is compared between controland treatment groups.

EXAMPLE 10 Apolipoprotein E and apoE-Mimetic Peptides Initiate aCalcium-Dependent Signaling Response in Macrophages

[0120] This example shows that apoE initiates a signaling cascade inmurine peritoneal macrophage that is associated with mobilization ofintracellular Ca²⁺ stores following increased production of inositoltrisphosphate. This cascade was inhibited by pretreatment withreceptor-associated protein and Ni²⁺. Signal transduction was mediatedby a pertussis toxin-sensitive G protein. These are characteristicproperties of signal transduction induced via ligand binding to thelipoprotein receptor-related protein (LRP) receptor. A peptide derivedfrom the receptor binding region of apoE also initiated signaltransduction in the same manner as the intact protein. The presence ofcross desensitization suggested that the apoE and the apoE-mimeticpeptide competed for the same binding site. This was confirmed by ourobservation that radiolabeled apoE-mimetic peptide competed with theintact protein for receptor binding. These data indicates thatApoE-dependent signal transduction mediates the immunomodulatoryproperties of this lipoprotein.

[0121] A. Materials and Methods

[0122] Materials. Brewer's thioglycollate broth was purchased from DifcoLaboratories (Baltimore, Md.). RPMI Medium 1640, fetal bovine serum,Hanks' Balanced Salt Solution and other cell culture reagents werepurchased from Life Technologies, Inc. (Grand Island, N.Y.). Bovineserum albumin (BSA), pertussis toxin, and HEPES were from Sigma ChemicalCo. (St. Louis, Mo.). Fura-2AM and BAPTA/AM were obtained from MolecularProbes (Eugene, Oreg.). Myo-[2-³H]inositol (specific activity 10-20Ci/mmol) was purchased from American Radiolabeled Biochemicals (St.Louis. Mo.). A plasmid containing the RAP cDNA was a kind gift from Dr.Joachim Herz, the University of Texas, Southwestern, Dallas Tex. It wasused to produce RAP as previously described [21]. Human recombinantapoE2 was obtained commercially from Panvera Corp (Madison, Wis.). Thepreparation was free of endotoxin, and homogenous as judged bySDS-polyacrylamide gel electrophoresis. [³H]thymidine (specificactivity, 70 Ci/mmol) and Iodine-125 (specific activity: 440 mCi/mg)were purchased from the American Radiolabeled Chemicals, Inc. (St. LouisMo.). The 20 amino acid ApoE mimetic peptide(Ac-TEELRVRLASHLRKLRKRLL-amide) with and without a tyrosine on the aminoterminus as well as a scrambled control peptide of identical size, aminoacid composition, and purity were synthesized by QCB Biochemicals(Hopkinton, Mass.) to a purity of 95%. All amino termini were acetylatedand all carboxyl termini were blocked with an amide moiety. Peptideswere reconstituted in sterile isotonic phosphate buffered saline. Ascrambled control peptide of identical size, amino acid composition, andpurity was also synthesized. All other reagents used were of the highestquality commercially available.

[0123] Macrophage Harvesting. All experiments involving animals werefirst approved by the Duke Institutional Animal Care and Use Committee.Pathogen-free female C57BL/6 mice and ApoE deficient mice previouslybackcrossed 10 times to the C57BL/6 strain were obtained from theJackson Laboratory (Bar Harbor, Me.). Thioglycollate-elicited peritonealmacrophages were harvested by peritoneal lavage using 10 ml of ice-coldHanks' balanced salt solution containing 10 mM HEPES and 3.5 mM NaHCO₃(HHBSS), pH 7.4. The macrophages were pelleted by centrifugation at 4°C. at ˜800×g for 10 min and resuspended in RPMI 1640 media supplementedwith 25 mM HEPES, 12.5 U/ml penicillin, 6.5 mg/ml streptomycin, and 5%fetal bovine serum. Cell viability was determined by the trypan blueexclusion method and was consistently greater then 95%.

[0124] Receptor Binding Studies. Macrophages were plated in 48-well cellculture plates (Costar) at 2.5×10⁵ cells per well and incubated for 3 hat 37° C. in a humidified 5% CO₂ incubator. The plates were then cooledto 4° C. and unbound cells were removed by three consecutive rinses withice-cold Hanks' balanced salt solution containing 20 mM Hepes and 5%BSA, pH 7.4 (binding buffer). To quantify direct binding of the¹²⁵I-apoE mimetic peptide, varying amounts of radiolabeled peptide wereadded to each well in the presence or absence of 200-fold molar excessof unlabeled peptide. Specific binding to cells was determined bysubtracting the amount of ¹²⁵I-apoE peptide bound in the presence ofexcess unlabeled peptide (nonspecific binding) from the amount of¹²⁵I-apoE peptide bound in the absence of excess unlabeled peptide(total binding). For competition studies, 50 nM radiolabeled peptide wasadded to each well in the presence or absence of varying amounts (31.25nM-4 □M) of unlabeled ApoE2 or RAP. Cells were then incubated at 4° C.for 12-16 h. Unbound ligand was removed from the wells and the cellmonolayer was rinsed three times with ice-cold binding buffer. Cellswere then solubilized with 1 M NaOH, 0.5% SDS at room temperature for >5h before the contents of each well was added to polystyrene tubes andcounted in a LKB-Wallac, CliniGamma 1272 □-counter (Finland).

[0125] Measurement of [Ca²⁺]_(i) in apoE and peptide treated macrophage.Changes in [Ca²⁺]_(i) levels in Fura-2/AM treated single cells werequantified using digital imaging microscopy in accordance with knowntechniques. Macrophages were plated on glass coverslips sitting in 35 mmPetri dishes at a density of 1.5×10⁵ cells/cm, and allowed to adhere for2 h in a humidified 5% CO₂ incubator at 37° C. The non-adherent cellswere aspirated and the monolayers were washed twice with HHBSS. 4 μMFura-2/AM was incubated with the cells for thirty min in the dark atroom temperature and [Ca²⁺]_(i) was subsequently measured using adigital imaging microscope in accordance with known techniques. Afterobtaining baseline measurements for 5 min, ligand (apoE, apoE mimeticpeptide, or scrambled peptide) was added, and multiple [Ca²⁺]_(i)measurements were taken. To determine if signaling resulted fromligation of the ligand to LRP, cells were preincubated with a 1000-foldmolar excess of RAP or 10 mM NiCl₂, both of which inhibit ligand bindingto LRP, for 5 min prior to stimulation with apoE or peptide. Inexperiments in which the involvement of a G protein was assessed,monolayers were incubated with 1 μg/ml pertussis toxin for 12 h at 37°C. and Ca²⁺ measurements were made as stated above.

[0126] Measurement of IP₃ in apoE treated macrophage and effect ofpertussis toxin. The formation of IP₃ in myo-[2-³H]inositol-labeledmacrophages under various experimental conditions was quantified inaccordance with known techniques. Macrophage were plated in 6 wellplates (4×10⁶ cells/well) and allowed to adhere at 37° C. for 2 h in ahumidified 5% CO₂ incubator. Medium was aspirated from the monolayersand RPMI 1640 medium containing 0.25% BSA and myo-[2-³H]inositol(specific activity 10-20 Ci/mmol) was added to each well. The cells wereincubated at 37 C. for an additional 16-18 h. Monolayers were rinsedthree times with 25 mM HHBSS containing 1 mM CaCl₂, 1 mM MgCl₂, 10 mMLiCl, pH 7.4. A volume of 0.5 ml of this solution was added to eachwell, and the cells were preincubated for 3 min at 37° C. beforestimulated with ligand. The reaction was stopped by aspirating themedium containing the ligand and adding 6.25% perchloric acid. The cellswere scraped out of the wells, transferred to tubes containing 1 ml ofoctylamine/Freon (1:1 vol/vol) and 5 mM EDTA, and were centrifuged at5600×g for 20 min at 4° C. The upper phase solution was applied to a 1ml Dowex resin column (AG1-X8 formate; Bio Rad Laboratories, Riclunond,Calif.) and eluted sequentially in batch process with H₂O, 50, 200, 400,800, and 1200 mM ammonium formate containing 0.1 M formic acid [26].Radioactivity was determined by placing aliquots in a liquidscintillation counter to determine radioactivity. To evaluate thepertussis-toxin sensitivity of the G protein coupled to receptoractivation and phosphatidyl inositol 4,5-bisphosphate (PIP₂) hydrolysis,cells were plated as described above and incubated with 1 μg/mlpertussis toxin which had been preactivated with 40 mM DTT at 30° C. for20 min. The effect on IP₃ formation was measured as described above.

[0127] Competition between apoE and apoE mimetic peptide for bindingsite on the receptor. Changes in macrophage [Ca²⁺]_(i) upon stimulationwith apoE and apoE-mimetic peptide were studied to determine whetherthese ligands bind to the same receptor. Fura-2/AM loaded macrophageswere incubated overnight, plated on glass cover slips, stimulated withone ligand, and changes in [Ca²⁺]_(i) quantified. Cells were thenstimulated with second ligand and Ca²⁺ measurements repeated

[0128] B. Results

[0129] Effect of apoE on macrophage [Ca²⁺]_(i). Modulation of freecytoplasmic Ca²⁺ concentration is a ubiquitous signaling response. Inmany cell types, binding of ligands to plasma membrane receptorsactivates the hydrolysis of PIP₂ by membrane-bound phospholipase C,generating IP₃. IP₃ causes the release of Ca²⁺ from the endoplasmicreticulum by binding to its cognate receptor, which is also a Ca²⁺channel. In non-excitable cells, [Ca²⁺]_(i) signaling is associated bothwith Ca²⁺ release from intracellular stores and Ca²⁺ influx. Treatmentof macrophages with human recombinant apoE increased [Ca²⁺]_(i) levels2-4-fold compared to macrophage treated with buffer (FIG. 8A). In atypical experiment [Ca²⁺]_(i) levels in unstimulated cells andapoE-treated cells were 95.33±7.37 and 180.25±14.57 nM, respectively.The increase in [Ca²⁺]_(i) upon stimulation with apoE was observed in70-80% of the cells examined. ApoE-induced increase in [Ca²⁺]_(i) washeterogeneous, asynchronous, and either oscillatory or sustained.ApoE-induced increases in macrophage [Ca²⁺]_(i) was dose-dependent (FIG.8B). To address the possibility that native apoE secreted by macrophagealtered responses to exogenous human recombinant apoE, these experimentswere repeated using macrophage prepared from apoE deficient mice.Calcium responses following stimulation with apoE were identical inwild-type macrophages and macrophages from apoE deficient mice (data notshown).

[0130] The effect of pertussis toxin on apoE-induced IP₃ synthesis.Exposure of myo-[2-³H] inositol-labeled macrophage to apoE caused a1.5-2.0-fold increase in IP₃ levels (FIG. 9A). This effect wasdose-dependent (FIG. 9B). Pretreatment of the macrophages with pertussistoxin completely abolished this increase in IP₃. (FIG. 9A). Thesestudies demonstrate that the phospholipase C-catalyzed hydrolysis ofmembrane PIP₂ in apoE stimulated cells is coupled to a pertussistoxin-sensitive G protein.

[0131] ApoE-induced increases in macrophage [Ca²⁺]_(i) are attenuated byNi²⁺ and RAP. Previous studies have demonstrated that ApoE binds to LRPand is then internalized. Additionally, binding of lactoferrin,Pseudomonas exotoxin A, lipoprotein lipase and thrombospondin to LRPinitiates a signaling cascade associated with the generation of secondmessengers. To investigate the possibility that LRP is involved in thesignal cascade induced by apoE, macrophages were preincubated with RAPand Ni⁺² prior to stimulation with apoE2 or apoE2 mimetic peptide. RAPis a 39 kD protein that blocks the binding of all known ligands to LRP.Ni⁺² also blocks ligand interactions with LRP. Both preincubation withRAP and Ni⁺² markedly attenuated the [Ca²⁺]_(i) increases associatedwith subsequent exposure to apoE (data not shown). These results areconsistent with the hypothesis that apoE induces a signaling cascade viaspecific interaction with LRP. Pretreatment of macrophage with pertussistoxin also markedly attenuated the ApoE-dependent Ca²⁺ response,indicating that signal transduction induced by apoE is coupled to apertussis toxin-sensitive G protein. This is consistent with the knownproperties of LRP-dependent signal transduction.

[0132] Effect of apoE-mimetic peptide on macrophage [Ca^(2+]) _(i).Stimulation of macrophage with the peptide derived from residues 130-149of the apoE receptor binding region also resulted in a 2-3-fold increasein [Ca²⁺]_(i) whereas a scrambled control peptide of identical size andcomposition had no effect (data not shown). This increase in [Ca²⁺]_(i)was observed in approximately 60-70% of cells examined. As with the apoEresponses, peptide-induced increases in macrophage [Ca^(2+]) _(i) wereheterogeneous and asynchronous. These results demonstrate that bothintact apoE and a peptide derive from the apoE receptor binding regioninduce an increase in [Ca²⁺]_(i) that is consistent with the initiationof a signaling cascade. However, on a molar basis, higher concentrationsof peptide were necessary to get [Ca²⁺]_(i) responses compared to theintact apoE. This difference likely results from differences in receptoraffinity between the peptide and apoE, a property generally seen whencomparing the effects of intact proteins to peptide ligands.

[0133] Effects of repeated stimulation of apoE and apoE-mimetic peptideon [Ca²⁺]_(i). We evaluated the possibility of competition between apoEand its mimetic peptide for binding sites on the receptor by quantifyingthe changes in [Ca²⁺]_(i) consequent to receptor ligation. Followingrepeated exposure to apoE, there was a marked attenuation in [Ca²⁺]_(i)suggesting tachyphylaxis (data not shown). Following the increase in[Ca²⁺]_(i) associated with the initial exposure to human recombinantapoE, there was a marked attenuation in [Ca²⁺]_(i) response tosubsequent peptide exposure (data not shown). Similarly, there was aloss of [Ca²⁺]_(i) response to apoE addition following initial exposureto peptide (data not shown). No desensitization in calcium response wasobserved with exposure of scrambled peptide (data not shown). Thisobserved tachyphylaxis suggests receptor desensitization secondary toreceptor ligation, and is consistent with the hypothesis that both theintact apoE protein and the 20 residue peptide bind to the samereceptor.

[0134] C. Discussion

[0135] The primary observations of this example are that: 1) binding toreceptors on the macrophage cell surface of human recombinant apoE (inpM to nM concentrations) initiates signaling events associated withincreases in [Ca²⁺]_(i) and IP₃; 2) a 20 residue peptide derived fromthe receptor binding region of apoE, but not a scrambled controlpeptide, causes identical changes in macrophage [Ca²⁺]_(i); 3) changesin [Ca²⁺]_(i) and IP₃ are specific and dose-dependent; 4) apoE-inducedincrease in cellular IP₃ is pertussis toxin-sensitive; and 5) changes in[Ca²⁺]_(i) are blocked by RAP and Ni²⁺. Moreover, based on the presenceof cross-desensitization, apoE and the apoE-mimetic peptide appear tobind to the same receptor.

EXAMPLE 11 An Apolipoprotein E Mimetic Peptide is Protective in a MurineHead Injury Model

[0136] This Example demonstrates a protective effect of intravenousadministration of a 17 amino acid apoE mimetic peptide (the fragment ofApoE containing amino acids 133-149) following head injury.

[0137] Mice were endotracheally intubated and their lungs weremechanically ventilated with 1.6% isoflurane at 30% partial pressure ofoxygen. The mice received a midline closed head injury delivered by apneumatic impactor at a speed of 6.8 m/s. Thirty minutes after closedhead injury, mice were randomized into 3 groups (n=16 mice per group asfollows: high dose peptide (406 ug/kg), low dose peptide (203 ug/kg),and saline control solution. All peptide solutions were prepared insterile isotonic saline (100 ul) and delivered intravenously via tailvein injection. Rotorod time and weight were measured for fiveconsecutive days after injury. At 21 days, the ability to learn to finda hidden platform in the Morris Water Maze was tested.

[0138] Prior to injury, rotorod latency and weights were comparable inall animals. After injury, the saline injected animals had a profounddeficit in rotorod testing which was associated with weight loss. Highdose peptide, and to a lesser extent low dose peptide protected animalsfrom this motor deficit (FIG. 10A), and concomitant weight loss (FIG.10b). This protective effect of the single dose of peptide was sustainedfor five days following injury (p<0.05 3-way repeat measures ANOVA).

[0139] In addition, the peptide appeared to provide protection inlearning deficits in learning to find a hidden platform (FIG. 10C) inthe Morris Water Maze (p<0.05 3-way repeat measures ANOVA). Treatmentwith the peptide also resulted in a significant improvement in acutesurvival as demonstrated by Kaplan-Meier analysis (FIG. 10D).

[0140] The foregoing examples are illustrative of the present invention,and are not to be construed as limiting thereof. The invention isdescribed by the following claims, with equivalents of the claims to beincluded therein.

1 9 1 16 PRT Artificial Sequence Description of Artificial Sequencefragment of ApoE containing LDL receptor binding site. 1 His Leu Arg LysLeu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu 1 5 10 15 2 9 PRTArtificial Sequence Description of Artificial Sequence fragment of ApoEcontaining LDL receptor binding site. 2 Leu Arg Lys Leu Arg Lys Arg LeuLeu 1 5 3 18 PRT Artificial Sequence Description of Artificial Sequencefragment of ApoE containing LDL receptor binding site. 3 Leu Arg Lys LeuArg Lys Arg Leu Leu Leu Arg Lys Leu Arg Lys Arg 1 5 10 15 Leu Leu 4 31PRT Artificial Sequence Description of Artificial Sequence fragment ofApoE containing LDL receptor binding site. 4 Thr Glu Glu Leu Arg Val ArgLeu Ala Ser His Leu Arg Lys Leu Arg 1 5 10 15 Lys Arg Leu Leu Arg AspAla Asp Asp Leu Gln Lys Arg Leu Ala 20 25 30 5 31 PRT ArtificialSequence Description of Artificial Sequence fragment of ApoE containingLDL receptor binding site. 5 Thr Glu Glu Leu Arg Val Arg Leu Ala Ser HisLeu Arg Lys Leu Arg 1 5 10 15 Lys Arg Leu Leu Arg Asp Ala Asp Asp LeuGln Lys Cys Leu Ala 20 25 30 6 20 PRT Artificial Sequence Description ofArtificial Sequence fragment of ApoE containing LDL receptor bindingsite. 6 Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu Arg1 5 10 15 Lys Arg Leu Leu 20 7 31 PRT Artificial Sequence Description ofArtificial Sequence fragment of ApoE containing LDL receptor bindingsite. 7 Thr Glu Glu Leu Arg Val Arg Leu Ala Arg His Leu Arg Lys Leu Arg1 5 10 15 Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala 2025 30 8 31 PRT Artificial Sequence Description of Artificial Sequencefragment of ApoE containing LDL receptor binding site. 8 Thr Glu Glu LeuArg Val Arg Leu Ala Arg His Leu Arg Lys Leu Arg 1 5 10 15 Lys Arg LeuLeu Arg Asp Ala Asp Asp Leu Gln Lys Cys Leu Ala 20 25 30 9 20 PRTArtificial Sequence Description of Artificial Sequence fragment of ApoEcontaining LDL receptor binding site. 9 Thr Glu Glu Leu Arg Val Arg LeuAla Arg His Leu Arg Lys Leu Arg 1 5 10 15 Lys Arg Leu Leu 20

That which is claimed is:
 1. A method of suppressing microglialactivation in a mammalian subject, comprising administering to the brainof said subject a compound that binds to microglial cells at thereceptor bound by a peptide of SEQ ID NO:3 (the LRP/α2M receptor), saidcompound administered in an amount effective to reduce microglialactivation compared to that which would occur in the absence of thecompound.
 2. A method of ameliorating symptoms associated with CNSinflammation in a subject, comprising administering to the brain of saidsubject a compound that binds to microglial cells at the receptor boundby a peptide of SEQ ID NO:3 (the LRP/α2M receptor), said compoundadministered in an amount effective to reduce microglial activationcompared to that which would occur in the absence of the compound.
 3. Amethod of ameliorating symptoms associated with CNS ischemia in asubject, comprising administering to the brain of said subject acompound that binds to microglial cells at the receptor bound by apeptide of SEQ ID NO:3 (the LRP/α2M receptor), said compoundadministered in an amount effective to reduce microglial activationcompared to that which would occur in the absence of the compound.
 4. Amethod according to any one of claims 1, 2 or 3, wherein said compoundis a peptide comprising the receptor binding domain of apolipoprotein E.5. A method according to any one of claims 1, 2 or 3 wherein saidcompound is a peptide comprising the amino acid sequence LRKLRKRLLLRKLRKRLL (SEQ ID NO: 3).
 6. A method according to any one of claims 1, 2or 3 wherein said compound is a dimer of two peptides, each peptidecomprising the amino acid sequence LRKLRKRLL (SEQ ID NO: 2).
 7. A methodaccording to any one of claims 1, 2 or 3 wherein said compound is apeptide of at least about 15 amino acids and comprises the amino acidsequence LRKLRKRLL (SEQ ID NO:2).
 8. A method according to any one ofclaims 1, 2 or 3, wherein said compound is a peptide comprising SEQ IDNO:6.
 9. A method according to claim 2, wherein said subject isafflicted with a condition selected from Alzheimer's disease andmultiple sclerosis.
 10. A method according to claim 3, wherein saidsubject is afflicted with a condition selected from stroke, globalcerebral ischemia, cerebral edema, closed acute head injury, andintracranial hemorrhage.
 11. A method according to any one of claims 1,2 or 3, wherein said compound is conjugated to a carrier molecule,wherein the presence of the carrier molecule increases transport of saidcompound across the blood-brain barrier, compared to that which wouldoccur in the absence of said carrier molecule.
 12. A method according toany one of claims 1, 2 or 3, wherein the mode of administration isselected from parenteral administration, intrathecal administration, andspinal administration.
 13. A method according to any one of claims 1, 2or 3, wherein said peptide is administered prophylactically prior toscheduled surgery.